The overwhelming majority of lung cancers are associated with smoking and most lung cancers express nicotinic acetylcholine receptors (nAChR) that are activated by the nicotine in cigarette smoke. The objective of this application is to characterize how the interaction of nicotine with nicotinic acetylcholine receptors (nAChR) expressed by lung cancers stimulates tumor growth with the ultimate objective of developing new therapeutic approaches to lung cancer by blocking this proliferative pathway. The nicotinic receptors are ligand-gated ion channels composed of 5 subunits, either a mixture of a and subunits or 5 of the same a subunit. Binding of a nicotinic agonist such as acetylcholine or nicotine opens the ion channel allowing entry of Na+ or Ca++ into the cell. While the nAChR are best known for their role as neurotransmitter receptors in the CNS, they are also widely expressed in non-neuronal cells. Our laboratory has been a leader in showing that essentially all lung cancers express nAChR and binding of nicotine to the nAChR stimulates lung cancer growth. The real world importance of nicotine as a stimulus for lung cancer growth has recently been confirmed by multiple genome-wide association studies linking polymorphisms in nicotinic receptors to increased risk of lung cancer even when corrected for numbers of cigarettes smoked. Strongest linkage by far has been with polymorphisms in the 15q25.1 nicotinic receptor gene cluster that encodes thea3, a5 and 4 nAChR subunits. Critically, the exact role of these nAChR subunits in mediating the ability of nicotine to stimulate cancer growth is unknown. In addition, how the polymorphism of greatest interest, rs16969968 which changes the Asp at residue 398 (a5D398) of the 15 nAChR subunit to Asn (a5N398) affects lung cancer growth is totally unknown. It is also likely that besides the a5D398 to a5N398 mutation, other mutations occur in the 15q25.1 nAChR gene locus that affects lung cancer growth. Therefore, we propose the following 3 specific aims: 1, To determine which nicotinic receptor subtypes in the a3, a5 and 4 nAChR gene locus are required for nicotine to stimulate lung cancer growth; 2, To determine the role of the a5D398 to a5N398 mutation (rs16969968) in mediating increased growth and development of lung cancer; and 3, To determine if there are additional common polymorphisms in the a3, a5 and 4 nAChR subunits that may increase lung cancer responses to nicotine. Because non-small cell lung cancer (NSCLC) is much more frequent than small cell lung carcinoma (SCLC) we will focus on NSCLC and specifically on squamous cell lung carcinoma (SCC) and lung adenocarcinoma (AC) which are the most frequent forms of NSCLC. These aims will be accomplished using a mix of cell biology, electrophysiology and in vivo studies in nude and transgenic mice to characterize actions of the nAChR receptors in lung cancer. From these studies will come understanding of which nAChR receptors mediate the effects of nicotine on lung cancer growth and thus identify which nAChR and proliferative pathways can be effectively targeted to develop new therapeutic approaches for lung cancer.